WO2002074972A1

WO2002074972A1 - Method of assaying lupus anticoagulant and assay reagent

Info

Publication number: WO2002074972A1
Application number: PCT/JP2002/002599
Authority: WO
Inventors: Tatsuya Atsumi; Takao Koike
Original assignee: Eisai C0. Ltd.
Priority date: 2001-03-19
Filing date: 2002-03-19
Publication date: 2002-09-26
Also published as: DE60222878D1; JPWO2002074972A1; EP1380653A4; US20040110242A1; JP4134300B2; US7244574B2; EP1380653A1; EP1380653B1; ATE375398T1

Abstract

A monoclonal antibody which reacts with prothrombin forming a complex with phosphatidylserine but not with prothrombin forming no complex with phosphatidylserine; and a method of assaying an activity of a test plasma of inhibiting blood coagulation which involves: (1) the step of assaying the inhibitory activity of the test plasma on blood coagulation reaction and its inhibitory activity on the blood coagulation reaction; and (2) the step of comparing the inhibitory activity of the test plasma with the inhibitory activity of the antibody.

Description

Specification

Lupus anticoagulant assay and reagents

The field of the present invention relates to a method and a reagent for measuring lupus anticoagulant (hereinafter referred to as LA) that appears accompanying antiphospholipid antibody syndrome. Background art

Antiphospholipid antibody syndrome (hereinafter referred to as APS) is an autoimmune disease in which autoantibodies called antiphospholipid antibodies are demonstrated in the blood as clinical symptoms of arterial and venous thrombosis, miscarriage and stillbirth. According to the definition of APS, proof of anti-phospholipid antibody (hereinafter aPL) is essential for diagnosis, but aPL is an extremely diverse group of autoantibodies and its detection is not always easy. The draft revised classification criteria for APS was drafted in 1999.

1). IgG or IgM ^ ₂ glycoprotein I-dependent anti-cardiolipin antibody

2). LA positive,

It was described.

However, LA testing is said to be one of the difficult tests for both clinicians and clinical examiners, and it is known that results vary greatly depending on the test method, reagent type, and sample condition.

LA stands for “in vitro phospholipid-dependent coagulation reaction (activated partial thromboplastin time (Triplett LA. Lupus 7 (Supple2) ₅ S18-22, 1998; hereinafter also referred to as aPTT)”, and violin coagulation time (Triplett LA. Lupus 7 (Supple2) ₃ S18-22, 1998; hereinafter also referred to as KCT), and immunity that inhibits the dilution rasel venom time (Triplett LA. Lupus 7 (Supple2), S18-22, 1998; hereinafter also referred to as dRVVT) It is defined as “globulin,” and guidelines for testing are provided by the Anti-phospholipid Antibody Standardization Committee of the International Society of Thrombosis and Hemostasis in 1995.

Various measures have been taken to increase sensitivity and specificity at each facility. It is difficult to determine the presence of LA by this method, and it is usually done using a combination of several methods. That is,

1) .aPTT, KCTs dRVVT to screen for prolonged phospholipid-dependent coagulation time,

2) Mixing test (addition of normal plasma) shows that this prolonged clotting time is due to the presence of inhibitors in the patient's plasma,

3) .Absorption neutralization test using impaired platelets and phospholipids confirms that this inhibitor is an antiphospholipid antibody.

It is a step.

Since LA suppresses phospholipid-dependent reactions, low phospholipid concentration is a condition for detecting LA with high sensitivity. However, even under these conditions, the selection and preparation of reagents and the setting of standard values must be performed at each facility, and the problem is that the results differ greatly between facilities. For this reason, LA is not generally measured quantitatively.

As for the indicators related to LA, only prothrombin (hereinafter also referred to as PS / PT) which forms a complex with phosphatidylserine in the patient's blood in Atsimi T et al., Arthritis Rheum 43: 1982-93, 2000 It has been reported that a reactive, phosphatidylserine-dependent anti-prothrombin antibody (hereinafter aPS / PT) shows a strong correlation with LA. However, the involvement of antibodies to blood coagulation factor / phospholipid complexes other than aPS / PT cannot be ruled out in LA, and whether aPT / PS, a monoclonal antibody that does not bind to these complexes, inhibits blood coagulation. It was not known whether it could be used as a LA standard. Disclosure of the invention

An object of the present invention is to establish a semi-quantitative LA measurement method using a standard antibody so that LA can be accurately measured, which has conventionally had a large difference between results at each facility. It is in.

The present inventors have demonstrated that the inhibitory activity on blood coagulation of patient sera containing autoantibodies against various blood coagulation factors / phospholipid complexes is typified by monoclonal aPS / PT. Based on the hypothesis that measurement can be performed, a mouse monoclonal aPS / PT is prepared, and the clotting activity is measured using this as a standard. I thought it might be. The present inventors have succeeded in producing a mouse monoclonal aPS / PT that does not react with prothrombin (hereinafter also referred to as PT) but reacts only with PS / PT. Inhibition of the reaction, the fact that the monoclonal antibody can be used as a standard antibody in LA measurement, and the fact that the use of the monoclonal antibody as a standard enables accurate or semi-quantitative LA measurement. did.

The present inventors named the produced hybridoma a 231D cell and the antibody produced as a 231D antibody.

That is, the present invention

1. A monoclonal antibody having an inhibitory activity on blood coagulation, which reacts with prothrombin complexed with phosphatidylserine and does not react with prothrombin not formed with phosphatidylserine.

2. The monoclonal antibody according to 1, wherein the monoclonal antibody is derived from a mouse.

3. The monoclonal antibody according to 1, wherein the monoclonal antibody is an antibody produced by 231D cells (FEM BP-7936).

4. (1) measuring the inhibitory activity of the test plasma on the blood coagulation reaction, and the inhibitory activity on the blood coagulation reaction of the antibody according to any one of 1 to 3,

(2). Compare the inhibitory activity of the test plasma with the inhibitory activity of the antibody described in any one of 1-3.

A method for measuring an activity of a test plasma that inhibits a blood coagulation reaction, comprising the steps of:

5.4 A method for measuring lupus anticoagulant in a test plasma, comprising measuring the inhibitory activity by the method described in 5.4, and determining the amount of lupus anticoagulant using the inhibitory activity as an index.

6. The method of measuring the inhibitory activity on the blood coagulation reaction can be any of measurement of prolongation of activated partial thromboplastin time, measurement of prolongation of kaolin coagulation time, measurement of prolongation of diluted Russell snake venom, or a combination thereof. The method according to 4, wherein 7. It is necessary to include the monoclonal antibody according to any one of 1 to 3 as a constituent reagent. A specialty reagent for measuring lupus anticoagulant.

8. (1), selecting antibody-producing cells that produce a monoclonal antibody that reacts with prothrombin complexed with phosphatidylserine,

(2). Furthermore, select antibody-producing cells that produce monoclonal antibodies that do not react with prothrombin not forming a complex with phosphatidylserine.

3. The method for producing the monoclonal antibody according to 1 or 2, comprising collecting a monoclonal antibody produced by the antibody-producing cell selected by the selection method including the step. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a standard curve of clotting time in aPTT using the 231D antibody as a standard.

FIG. 2 shows the distribution of ACU in the APS group and the non-APS group measured using the 231D antibody as a standard. In the figure, the dotted line indicates the ACU reference value (mean of 30 healthy persons + 2SD). BEST MODE FOR CARRYING OUT THE INVENTION

The antibody of the present invention reacts with prothrombin (PS / PT) complexed with phosphatidylserine and does not react with prothrombin (PT) not complexed with phosphatidylserine.モノクローナル有するモノクローナルモノクローナルモノクローナルモノクローナル

In the present invention, “reacting with an antibody” means reacting immunologically. Also, a complex of phosphatidylserine (PS) and prothrombin (ΡΤ) forms a complex between PS and で under conditions close to physiological conditions (eg, pH 7.5, PS: PS = 3: 1 (weight ratio)). It refers to the complex that forms. The inhibitory activity on the blood coagulation reaction can be measured based on a change in a value used as an indicator of the blood coagulation reaction when the monoclonal antibody is present in the reaction system. Such values include aPTT, dRWT, KCT, and the like.

The monoclonal antibody of the present invention is preferably derived from a mouse.

The monoclonal antibody of the present invention is obtained by collecting lymphocytes from an animal immunized with human prothrombin (even humans having autoantibodies to human thrombin are allowed) and immortalizing them. Produce antibodies that react but do not react with PT The clones (antibody-producing cells) to be produced are selected, and the antibodies produced by the clones are collected.

The method of immortalization is not particularly limited, and it can be carried out by a method such as infecting with a virus (EBV for human cells) or cell fusion.

The method for selecting antibody-producing cells is not particularly limited, as long as an antibody that reacts with PS / PT but does not react with PT is selected. Antibody-producing cells include (1) monoclonal antibodies that react with PS / PT. It is preferable to select an antibody-producing cell that produces an antibody, and (2) to select an antibody-producing cell that produces a monoclonal antibody that does not react with PT.

Antibodies that react with PS / PT but not with PT usually have an inhibitory activity on the blood coagulation reaction, but should be confirmed if necessary.

The method of collecting the antibody produced by the antibody-producing cells is not particularly limited, and monoclonal antibodies are produced by cell culture, mouse ascites, etc., and the produced monoclonal antibodies are usually used for antibody purification. What is necessary is just to refine with the method used for.

Hereinafter, a method for producing a mouse monoclonal aPS / PT will be specifically described as an example, but the present invention is not limited thereto.

Purified or partially purified human prothrombin or human plasma is subjected to an appropriate treatment to enhance immunogenicity, if necessary, for example, a treatment such as making Freund's complete adjuvant or an emulsion with Freund's complete adjuvant. Immunize mice by subcutaneously, intraperitoneally, or footpad. If necessary, repeat the immunization several times at appropriate intervals.

From the immunized mouse, an organ containing antibody-producing cells such as spleen and lymph node is removed, and lymphocytes are separated therefrom through a mesh. Cell fusion between the isolated lymphocytes and myeloma cells, for example, P3U1 cells, is performed by the following method, but the cell fusion method is not limited to this.

Wash lymphocytes and P3U1 cells twice with RPMI-1640 medium, mix at a ratio of 5: 1 to 10: 1, suspend in HPMI-1640 medium, and centrifuge at 1500 rpm for 5 minutes. After removing the cell mass, slowly add 1 ml of 50% PEG solution (manufactured by Behringer) warmed at 37 ° C over 2 minutes. Then, gradually add 20 ml of RPMI-1640 medium warmed to 37 ° C over 3 minutes. Centrifuge at 1000 rpm for 5 minutes and leave for 5 minutes.

After removing the supernatant, suspend the cells in 10% FCS / RPMI-1640 medium so that the lymphocyte cells become 1 x 10 ⁶ / ml, and dispense 100 1 per well into a 96-well plate. On the next day, add 10% FCS / 2x HAT (manufactured by Lifetech) / RPMI-1640 medium at a ratio of 100% per 1 ml and perform hybridoma HAT selection to select the fused cells. In order to assist the growth of hybridomas, it is preferable to culture them with thymocytes of syngeneic mice or to add, for example, 1/10 volume of ORIGEN HCF (manufactured by ICGS). Culture is continued for an appropriate number of days, preferably for 1 to 2 weeks, and the culture supernatant is used for antibody assay. From the obtained clones, a clone that produces an antibody that reacts with PS / PT but does not react with PT is selected. The reaction with PS / PT or PT may be performed in the liquid phase, but the case where it is performed in the solid phase where the procedure is easy will be described below.

First, a method is described in which PS / PT is formed on a solid phase and clones that react with PS / PT are identified. After dissolving phosphatidylserine in an appropriate concentration, for example, 50 ig / ml in a solvent, for example, methanol nocroform, and adding the solution to an ELISA plate (manufactured by Sumitomo Beichik Co.), the solvent is evaporated. Thereafter, in order to prevent the antibody from non-specifically adsorbing to the ELISA plate, a buffer solution containing a protein such as albumin 'casein (skim milk)' gelatin and a suitable concentration of, for example, 5 mM Ca ²⁺ (blocking solution). Solution) to the ELISA plate and perform blocking.

After washing, a prothrombin solution containing human prothrombin in a suitable concentration, for example, 10 / g / ml, is added to the ELISA plate. The added human prothrombin binds to phosphatidylserine via the gla region in the presence of Ca ^{2 +} to form PS / PT with an altered conformation. As a control, prepare a well containing a blocking solution without human prothrombin.

The collected hybridoma culture supernatant is added to an ELISA plate on which PS / PT has been formed, and reacted. After washing, an appropriate label, for example, an enzyme label such as alkaline phosphatase 'peroxidase, a fluorescent label, or a radioactively labeled anti-mouse Ig antibody is added and reacted. After washing, a method appropriate for the label is used. If the label is an enzyme, the enzyme substrate is added to develop the color. If the label is fluorescent, the fluorescence is measured. If the label is radioactive, the radioactivity is measured. Clones corresponding to pegs that had human prothrombin and had more labeling observed than pegs that did not contain human prothrombin were used as clones producing antibodies that react with PS / PT. Identify.

The buffer used for washing preferably contains an appropriate concentration, for example, 5 mM Ca ²⁺ , and an appropriate surfactant, for example, 0.05% Tween 20.

Subsequently, a method for selecting a clone that produces an antibody that does not react with PT will be described. After adding an appropriate concentration of human prothrombin to the ELISA plate, for example, 10 µg / ml, and washing, albumin 'casein (skim milk) to prevent antibodies from nonspecifically adsorbing to the ELISA plate 'Add a buffer containing a protein such as gelatin to the ELISA plate and perform blocking.

The hybridoma culture supernatant that has reacted with PS / PT is added to an EUSA plate and reacted. After washing, an appropriate label, for example, an enzyme label such as alkaline phosphatase / peroxidase, a fluorescent label, or a radioactively labeled anti-mouse Ig antibody is added and reacted. After washing, a method appropriate for the label is used. If the label is an enzyme, the enzyme substrate is added to develop the color. If the label is fluorescent, the fluorescence is measured. If the label is radioactive, the radioactivity is measured. The clone corresponding to the unlabeled mouse is identified as a clone producing an antibody that does not react with PT.

As mentioned above, immunization of mammals, acquisition of antiserum, production of hybridomas, enzymatic hyperactivity, method {3 literature, f row _ Pama Ed Harlow, David Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

The antibody was purified from the culture supernatant obtained by culturing the cells of the obtained clones, or from the mouse ascites inoculated with the cells of the clones, preferably using a protein A column or the like, to confirm that the purified antibodies have coagulation inhibitory activity. , DRVVT and KCT are measured and examined.

The measurement is performed using, for example, PTT-LA (Diagnostica Stago) for aPTT, dRVVT (Gradipore LA, MBL) Reagent 1 for dRVVT, and kaolin (Behringer) for KCT, for example. PTT-LA and dRVVT measure clotting time according to the package insert. In addition, KCT mixes the standard or sample and the kaolin reagent in a 50〃1 mixture, incubate at 37 ° C for 2 minutes, and add 50 zl of 0.025 M CaCl ₂ to increase the coagulation time. Measure.

Confirm that the obtained antibodies prolong clotting time in a dose-dependent manner.

The 231D antibody according to one embodiment of the present invention can be obtained from 231D cells. The 231D cells were obtained on March 9, 2001, by the National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary, Yuichi (former name: National Institute of Advanced Industrial Science and Technology), postal code: 305-8 566 Deposited at 1-chome, 1-chome, Tsukuba, Ibaraki Prefecture, Japan No. 1 Central No. 6) under accession number FERM P-182 47, transferred to an international deposit based on the Budapest Treaty on March 5, 2002, accession number ίΈ BP -7936 has been granted.

The antibody of the present invention can be used as a standard when measuring LA. Therefore, the present invention provides a method for measuring LA using the antibody of the present invention as a standard.

The method for measuring LA of the present invention is a method for measuring the activity of a test plasma to inhibit the blood coagulation reaction, which comprises the following steps: (1) the inhibitory activity of the test plasma on the blood coagulation reaction; Measuring the inhibitory activity of the body on the blood coagulation reaction,

(2). Compare the inhibitory activity of the test plasma with the inhibitory activity of the antibody of the present invention

A process.

The method of measuring the inhibitory activity on the blood coagulation reaction is not particularly limited, and a substance whose inhibitory activity is to be measured is added to a reaction solution for measuring the blood coagulation reaction, and the blood coagulation reaction is performed. The inhibitory activity can be measured by measuring the effect on the reaction.

The method of measuring the inhibitory activity on the blood coagulation reaction is preferably any one of measurement of prolongation of activated partial thromboplastin time, measurement of prolongation of kaolin coagulation time, measurement of prolongation of diluted Russell snake venom time, or It is a combination.

For example, when the inhibitory activity on the blood coagulation reaction is measured by measuring the prolongation of the coagulation time, a standard sample containing various concentrations of the antibody of the present invention is prepared, and the coagulation time of the standard sample is measured. Is plotted on the Y axis and the concentration of the antibody of the present invention is plotted on the X axis to prepare a standard curve, and the coagulation time of the test plasma is plotted on the standard curve.

The amount of lupus anticoagulant in the test plasma is measured by determining the amount of lupus anticoagulant using the inhibitory activity measured by the above method as an index. Can be specified.

Hereinafter, a method for measuring LA using mouse monoclonal aPS / PT as a standard will be specifically described as an example, but the present invention is not limited thereto.

Platelets are removed by passing normal plasma through a 0.22 / m filter, and the platelet-depleted plasma is treated with an appropriate serial dilution of purified 231D antibody, e.g., a 2-fold dilution series of 50-1.3 g / ml. And add 0 g / ml as the standard sample.

The platelet is also removed from the patient's plasma through a 0.22 m filter, and the platelet-depleted patient's plasma and the platelet-removed normal plasma are also added in an appropriate ratio, for example, patient plasma: normal plasma = 20:80 (Volume ratio) to obtain the test plasma.

The coagulation time of the standard sample and the test plasma is measured. For the reagent, aPTT uses, for example, PTT-LA (Diagnostica Stago), dRWT uses, for example, dRVVT (Gradipore LA, MBL) Reagent 1, and KCT uses, for example, kaolin (Beilinger). PTT-LA and dRVVT measure clotting time according to the package insert. For KCT, mix the standard or sample with kaolin reagent in 50〃1 mixture, incubate for 2 minutes at 37 ° C, then add 50〃1 of 0.025 M CaCl ₂ and measure the coagulation time.

For each reagent, create a standard curve by plotting the clotting time of the standard sample on the Y-axis and the concentration of 231D antibody on the X-axis. Put the coagulation time of the test plasma on a standard curve, read how many g / ml of the anticoagulant activity of the test plasma corresponds to the 231D antibody, and use it as the ACU (Anticoagulant Unit) of the test plasma. Expressed as In addition, check the ACU of ten healthy people with each reagent, and set the standard value on average + 2SD.

Test sera showing an ACU greater than the reference value are judged to be LA-positive.

The present invention further provides a reagent for measuring Rubes anticoagulant, which comprises the antibody of the present invention as a constituent reagent.

The reagent of the present invention may have the same configuration as a usual reagent for measuring Rubus anticoagulant except that it contains the antibody of the present invention used as a standard. The antibody of the present invention may be made into a composition by combining with a carrier (such as a buffer solution) that is acceptable as a reagent. Example

Hereinafter, the present invention will be described with specific examples, but the present invention is not limited thereto.

[Example 1] Preparation of mouse monoclonal aPS / PT

(1) Production of Hypri-Doma

Balb / c mice were immunized twice with 50 ngl of purified human prothrombin (manufactured by Enzym Research) together with complete Freund's adjuvant, and 14 days and 28 days later with incomplete Freund's adjuvant. After confirming that the antibody titer against human prothrombin had increased, the spleen was removed 42 days after the final immunization. After shredding the spleen, lymphocytes were separated through a mesh.

The separated lymphocytes and myeloma P3U1 cells were mixed at a ratio of 5: 1 to 10: 1, suspended in RPMI-1640 medium, and centrifuged. After loosening the cell mass, 1 ml of a 50% PEG solution (manufactured by Boehringer) warmed at 37 ° C was gradually added over 2 minutes. Subsequently, 20 ml of RPMI-1640 medium warmed to 37 ° C. was gradually added over 3 minutes. After centrifugation at 1000 rpm for 5 minutes, the cells were allowed to stand for 5 minutes to perform cell fusion. After removing the supernatant, the lymphocytes were suspended in a 10% FCS / RPMI-1640 medium so as to have a concentration of 1 × 10 ^s / ml, and the suspension was dispensed at 100 zl / well into 20 96-well plates. On the next day, 10% FCS / 2χ (manufactured by Lifetech) / RPMI-1640 medium was added at a ratio of 100 per 1 ml, and selection of hybridomas was started. The culture supernatant was collected 12 days after the start of the culture and used for antibody analysis.

(2). Selection of clones producing antibodies that react with PS / PT

Dissolve phosphatidylserine (manufactured by Sigma) at a concentration of 50 ig / ml in a solvent of methanol Z-cloth form, add 30〃1 of the solution to an ELISA plate (manufactured by Sumitomo Bei-Client), evaporate the solvent, and add albumin. A solution (10 mg / ml BSA (fatty acid free, manufactured by Sigma), 5 mM CaCl ₂ , 150 mM NaCl, 10 mM Tris-HCl pH 7.5) 150-1 was added and blocking was performed. After washing, add 50 プロ 1 of prothrombin solution (10〃g / ml human prothrombin (Diagonostica Stago)), 10 mg / ml BSA ヽ 5 mM CaCl ₂ , 150 mM NaCl, 10 mM Tris-HCl pH 7.5) and ELISA A PS / PT complex was formed on the plate. As a control, a permeate containing an albumin solution without human prothrombin was added. Was also prepared.

After washing the ELISA plate, 50-1 of the hybridoma culture supernatant diluted x20-xl, 000 was added to react with the PS / PT complex formed on the ELISA plate. After washing, the plate was further reacted with an anti-mouse Ig antibody labeled with alkaline phosphatase. After washing, a substrate of alkaline phosphatase was added to develop color. Compared with the well containing the albumin solution, the clone containing the purified human prothrombin solution and having a stronger color development was selected as a clone producing an antibody that reacts with PS / PT.

(3) Selection of clones producing antibodies that do not react with ΡΤ

To the ELISA plate, 80 μl of a 10 μg / ml purified human prothrombin solution dissolved in PBS was added, and after washing, a 0.5% gelatin solution was added to perform blocking.

After washing the ELISA plate, the hybridoma culture supernatant selected in (2) was diluted x20-xl, 000, added to 50 l, and reacted with PT on the ELISA plate. After washing, the cells were further reacted with an anti-mouse Ig antibody labeled with alkaline phosphatase. After washing, the plate was developed by adding a substrate for alkaline phosphatase, and the clone that did not develop was selected as a clone that produced an antibody that did not react with PT.

231D cells (FERM BP-7936) were selected as clones producing antibodies that reacted with PS / PT but did not react with PT. Hereinafter, the antibody produced by 231D cells is referred to as 231D antibody.

(4) Anticoagulant activity of .231D antibody

Platelet is removed from normal plasma through a 0.22 m filter, and purified 231D antibody is added to the platelet-depleted plasma to a concentration of 50, 25, 12.5, 6.3, 3.1, 1.3, Og / ml. The clotting time was measured.

The reagents used were PTT-LA (Diagnostica Stago Co.), Zeon T (Gradipore LA, MBL) reagent 1, and kaolin (Beilinger). PTT-LA and dRVVT measured clotting time according to the package insert. The KCT was prepared by mixing 50 or one of the standard or sample and the Kolin reagent at room temperature, incubating for 2 minutes at 37 ° C, and then adding 50〃1 of 0.025 M CaCl ₂ to measure the coagulation time.

Figure 1 shows the relationship between the amount of 231D antibody and the clotting time in a typical aPPT. 231D antibody Showed a dose-dependent prolongation of coagulation time in each coagulation system. These results suggested that the 231D antibody could be used as a standard for LA measurement.

[Example 2] LA measurement using 231D antibody as standard

(1). LA measurement method

From the results of Example 1 (4), the anticoagulant activity of the test plasma corresponding to 1 g / ml of the 231D antibody was defined as 1.0 ACU (Anticoagulant Unit) based on the standard curve prepared by serial dilution of the 231D antibody. Semi-quantitative measurements were made. Specifically, measurements were made as follows o

Platelets were removed from normal plasma through a 0.22 zm filter, and purified 231D antibody was added to the platelet-removed plasma to 50, 25, 12.5, 6.3, 3.1, 1.3, 0, and 0 g / ml to obtain a standard sample. . Similarly, the platelet is removed from the patient's plasma through a 0.22 / m filter, and the platelet-removed patient's plasma and the platelet-removed normal plasma are mixed at a patient plasma: normal plasma = 20:80 (volume ratio). And used as test plasma.

The coagulation time of the standard sample and the test plasma was measured. The reagents used were PTT-LA (Diagnostica Stago), dRVVT (Gradipore LA, MBL) Reagent 1, and kaolin (Beilinger). PTT-LA and dRVVT measured clotting time according to the package insert. The KCT was prepared by mixing the standard or sample and the violin reagent in 50 zl portions and incubating at 37 ° C for 2 minutes, then adding 50〃1 of 0.025 M CaCl ₂ and measuring the coagulation time.

For each reagent, a standard curve was prepared by plotting the clotting time of the standard sample on the Y-axis and the concentration of 231D antibody on the X-axis. The coagulation time of the test plasma was plotted on a standard curve, and the anticoagulant activity of the test plasma was read to what g / ml of the 231D antibody, and this was expressed as the ACU of the test plasma. In addition, the ACU of 10 healthy persons was examined for each reagent, and the standard value was set at an average of +2 SD.

(2). Measurement of LA in APS patients

Arterial and venous thrombosis or miscarriage 47 patients (APS group and abbreviated as T) and 116 patients without thrombosis or abortion (non-APS group and abbreviated as non-T) 163 in total LA was measured in immunologically ill patients. Those whose ACU exceeded the reference value (mean + 2SD for 30 healthy subjects) were 17/47 (36%) at T for aPTT alone and 12/116 (10%) for non-T . DRVVT alone was 13/47 (28%) in Τ and 13/116 (11%) in non-T. The KCT alone was 21/47 (45%) for Τ and 25/116 (22%) for ηοη-Τ (Fig. 2).

When the three types of aPTT, dRVVT and KCT were combined, those in all of which exceeded the ACU standard value (mean of 2 healthy subjects + 2SD) were 22 out of 47 in T (APS group), The percentage of -T (non-APS group) exceeding the reference value was 31 out of 116 patients, which was significantly higher in patients with APS.

(3) Influence of p-phalin on LA measurement

The gladipore reagent currently used for LA measurement is known to give false-positive cases when using monophalin. Therefore, the effect of perfarin on the LA measurement method using the 231D antibody as a standard was examined.

As a result, there was no particular effect when using plasma of a patient receiving p-farin (Table 1). table 1

aPTT (ACU) dRVVT (ACV) KCT (ACU)

1 30 20 30

2> 50> 50> 50

LA positive 3 35 17 34

4 48> 50> 50

5 30> 50> 50

6 15 14 41

7 <3.1 14 3.3

8 <3.1 12 <3.1

ヮ Use of monophalin 9 <3.1 3.5 <3.1

10 <3.1 3.5 <3.1

11 3.1 <3.1 <3.1

12> 50 43> 50

LA positive and

Use of perferin

From the above results, it was clarified that the use of the 231D antibody enables semi-quantitative measurement of LA and more accurate determination of LA. Industrial applicability

The fact that a monoclonal antibody was prepared as a standard for LA, and that the use of the monoclonal antibody as a standard made it possible to accurately and semi-quantitatively measure LA where there was a large difference between the facilities. Can be implemented.

Claims

The scope of the claims

1. A monoclonal antibody having an inhibitory activity on blood coagulation, which reacts with prothrombin complexed with phosphatidylserine and does not react with prothrombin not complexed with phosphatidylserine.

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is derived from a mouse.

3. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is an antibody produced by 231D cells (FERM BP-7936).

4. (1) measuring the inhibitory activity of the test plasma on the blood coagulation reaction, and the inhibitory activity on the blood coagulation reaction of the antibody according to any one of claims 1 to 4,

(2) comparing the inhibitory activity of the test plasma with the inhibitory activity of the antibody according to any one of claims 1 to 3

A method for measuring an activity of a test plasma, which inhibits a blood coagulation reaction, comprising the steps of:

5. A method for measuring lupus anticoagulant in a test plasma, comprising measuring the inhibitory activity by the method according to claim 4, and determining the amount of lupus anticoagulant using the inhibitory activity as an index.

6. The method of measuring the inhibitory activity on the blood coagulation reaction is to measure the prolongation of the activated partial thromboplastin time, the prolongation of the kaolin coagulation time, or the prolongation of the diluted Russell snake venom time, or a combination thereof. A method according to claim 4.

7. A reagent for measuring lupus anticoagulant, comprising the monoclonal antibody according to any one of claims 1 to 3 as a constituent reagent.

8. (1). Select antibody-producing cells that produce monoclonal antibodies that react with prothrombin complexed with phosphatidylserine,

The method for producing a monoclonal antibody according to claim 1 or 2, comprising collecting a monoclonal antibody produced by an antibody-producing cell selected by a selection method comprising a step.